Biological glue based on thrombin-conjugated nanoparticles

ABSTRACT

Thrombin-conjugated nanoparticles, wherein said nanoparticles comprise one or more organic and/or inorganic compounds and process for preparing the same are provided. The thrombin-conjugated nanoparticles are suitable for use in the preparation of fibrin-based biological sealant.

FIELD OF THE INVENTION

The present invention relates to thrombin-conjugated nanoparticles andformulations comprising the same, which are suitable for the preparationof fibrin-based biological glue.

BACKGROUND OF THE INVENTION

The art has recognized the potential of nanoparticles, which aregenerally defined as spherical particles having sizes ranging fromapproximately a few nanometers up to a few microns, for use in differentapplications in chemistry, biology and medicine. The preparation ofvarious nanoparticles and uses thereof are described in the followingpublications: Scientific and Clinical Applications of Magnetic Carriers,Ed. U. Hafeli, W. Schutt, J. Teller and M. Zborowski, Plenum Press, N.Y.37-51, 53-67 and 135-148 (1997); Recent Res. Developments inPolymerization Science, Ed. S. G. Patidalai, Transworld Research Network1, 51-78 (1997); O. Melamed and S. Margel, J. of Colloid and InterfaceScience 241, 357 (2001); V. Bendikiene and B. Juodka, Stability andStabilization of Biocatalysts, Ed. A. Ballesteros, F. J. Plou, J. L.Iborra and P. J. Halling, Elsevier Science 583-588 (1998) and J. Parradoand J. Bautista, Biosci Biotech. Biochem. 59 (5), 906 (1995).

The blood clotting process involves the cleavage of the plasma highlysoluble molecule fibrinogen by the action of the proteolytic enzymethrombin, following which the monomers obtained associate together toform fibrin, in the form of long, insoluble fibers. The formation of theinsoluble fibrin matrix may be accelerated by the presence of factorXIII and/or the presence of Ca⁺² ions. Thrombin converts factor XIII tofactor XIIIa, which, in the presence of CaCl₂, crosslinks the fibrinmatrix to give a highly crosslinked polymer.

The art proposed various formulations, known as fibrin glues, or fibrinsealants, in order to allow effective and rapid interaction betweenfibrinogen and thrombin, and devices useful for delivering saidformulations to the bleeding site, thereby mimicking the final step ofblood clotting process resulting in the production of the desired fibrinclot.

For example, the commercially available product Tisseel® Fibrin Sealant(Immuno AG, Austria) is based on a two compartments syringe forseparately holding a thrombin solution and fibrinogen solution. In use,the two components are simultaneously mixed and applied on the bleedingarea to form the insoluble fibrin glue matrix.

Another commercially available product, Quixil® (OmrixBiopharmaceuticals, Belgium) is based on one-compartment syringe, orsimilar arrangement, composed of a thrombin solution only. The thrombinsolution may also contain other components, e.g. antifibrinolytic agentssuch as aprotinin or tranexamic acid and/or factor XIII and/or CaCl₂.The thrombin solution is then sprayed, or compressed, or applied, on thebleeding area to form the insoluble fibrin by interacting with thefibrinogen content of the blood.

TachoComb® (NYCOMED GmbH, Munchen), is prepared by covering a sheet ofcollagen with human fibrinogen and bovine thrombin. Aprotinin is addedto prevent early degradation of the fibrin clot by plasmin. For thispurpose, the solid components: fibrinogen, thrombin and aprotinin aredispersed in an organic medium, and the suspension is applied on a sheetof collagen. The organic medium is evaporated, leaving a dried layer ofthe components of fibrin glue adsorbed on the collagen surface. When thecoating comes in contact with blood or other liquids, the componentsdissolve and fibrin is formed.

A. Sugitachi et al. [Progress in Artificial Organs, Ed. Y. Nose, C.Kjellstrand and P. Ivanovich, 1020-1022 (1985)] describe dry fine flakescontaining thrombin, gelatin and factor XIII, suitable for treatinghemostasis.

Horak et al. [Polymers in Medicine 21 (1-2), 31 (1991)] describe athrombin-containing hydrogel, based on poly(2-hydroxyethylmethacrylate), that is useful for endovascular occlusion.

Liu et al. [Analytical Biochemistry 147, 49 (1985)], describethrombin-gold nanoparticles, that are prepared by physical adsorption ofa monolayer of thrombin onto gold nanoparticles of 16.5±1.8 nm diameter.

It is a purpose of the present invention to provide a novel thrombinformulation and therapeutic compositions based thereon, that may be usedin the preparation of a fibrin sealant.

It is another purpose of the invention to provide a thrombin formulationthat is highly stable and easily deliverable, and a process forpreparing the same.

It is a further purpose of the invention to provide a thrombinformulation that permits rapid formation of the fibrin clot.

Further objects and advantages of the present invention will becomeapparent as the description proceeds.

SUMMARY OF THE INVENTION

The present invention is primarily directed to thrombin-conjugatednanoparticles, wherein said nanoparticles comprise one or more organicand/or inorganic compounds.

The term “thrombin-conjugated nanoparticles”, as used herein, refers tonanoparticles to which thrombin molecules are linked, either chemically(e.g., covalently), or by means of physical adsorption.

The nanoparticles comprise organic compounds, wherein said organiccompounds are preferably selected from the group consisting of proteinsand synthetic polymers, and/or inorganic compounds, wherein saidinorganic compounds are preferably selected from the group consisting ofmetal oxides or oxides of metalloids.

Preferably, the nanoparticles are selected from the group consisting ofiron oxide-containing nanoparticles, albumin nanoparticles, solid orhollow silicon oxide nanoparticles and nanoparticles made of organicpolymeric core coated with at least one silica shell, optionally havinga magnetic layer interposed between said core and said silica shell.Preferably, the average diameter of the thrombin-conjugatednanoparticles is not greater than 5 μm.

According to one preferred embodiment, the invention providesthrombin-conjugated nanoparticles, wherein the thrombin molecules arecovalently-bonded to the surface of the nanoparticles.

According to another preferred embodiment, the invention providesthrombin-conjugated nanoparticles, wherein the thrombin molecules arecovalently-bonded to spacer arms, and wherein said spacer arms arecovalently-linked to the surface of the nanoparticles. Most preferably,the spacer arm is albumin.

According to another preferred embodiment, the invention providesthrombin-conjugated nanoparticles, wherein the thrombin molecules arephysically adsorbed onto spacer arms, and wherein said spacer arms arecovalently-linked to the surface of the nanoparticles. Most preferably,the spacer arm is albumin.

Optionally, the thrombin-conjugated nanoparticles according to theinvention may further comprise a pharmaceutical agent which is eitherencapsulated within said nanoparticles or bound thereto.

In another aspect, the present invention relates to a process forpreparing thrombin-conjugated nanoparticles, comprising providingnanoparticles having reactive chemical groups on their surface, andeither covalently linking thrombin thereto, or covalently linking spacerarms to said reactive chemical groups and subsequently allowing thrombinmolecules to chemically react with said spacer arms, or to becomephysically adsorbed thereto. Preferably, the reactive chemical groupsare either activated carbon-carbon double bonds or aldehyde groups.Preferably, the spacer arm is albumin.

In another aspect, the present invention provides a therapeuticcomposition comprising thrombin-conjugated nanoparticles, suitable foruse in the preparation of fibrin-based biological sealant. According toone variant, the therapeutic composition is provided in the form of adry powder comprising nanoparticles, to which thrombin is conjugated,and a dispersant, which is most preferably gelatin. It has beensurprisingly found that such a formulation exhibits excellent storagestability and may be effectively used in the preparation of fibrin-basedbiological sealant. In another variant, the therapeutic composition isprovided in the form of a liquid suspension comprising thethrombin-conjugated nanoparticles.

In another aspect, the present invention provides a process forpreparing a thrombin formulation provided in the form of a dry powdercomprising thrombin-conjugated nanoparticles, wherein said processcomprises providing an aqueous suspension of said thrombin-conjugatednanoparticles and drying the same in the presence of a suitabledispersant, which is preferably gelatin. Preferably, the drying isaccomplished by means of lyophilization.

In another aspect, the present invention provides a method for preparinga fibrin-containing biological sealant, wherein said method comprisescontacting thrombin-conjugated nanoparticles, which preferably have anaverage diameter smaller than 5μ, with fibrinogen. Preferably, thethrombin conjugated nanoparticles and fibrinogen are contacted in aliquid medium selected from the group consisting of aqueous solution,plasma or blood, whereby the fibrin sealant is formed. According to apreferred embodiment of the invention, the thrombin-conjugatednanoparticles and fibrinogen are contacted in the presence of calciumions or factor XIII.

The fibrin biological sealant formed according to the present inventionis typically characterized by the presence of nanoparticles, the averagediameter of which being preferably smaller than 5 μm, within itspolymeric matrix.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Effect of the inhibitor Antithrombin III on the activity of Thrand immobilized Thr: SiO₂˜Thr (2.3 μm) (A), AB˜Thr (B) and Mag˜Thr (C)suspended in PBS.

FIG. 2. Clotting time in plasma as a function of storage at RT of Thrand SiO₂˜Thr suspended in PBS.

FIG. 3. Clotting time in plasma as a function of storage at RT of Thrand AB˜Thr suspended in PBS.

FIG. 4. Clotting time in plasma as a function of storage time at RT ofThr and Mag˜Thr suspended in PBS.

FIG. 5. Effect of sterilization on the clotting time in plasma of Thrand Mag˜Thr suspended in PBS and stored RT.

FIG. 6. Effect of sterilization on the clotting time in blood of Thr andMag˜Thr suspended in PBS and stored at RT.

FIG. 7. Effect of CaCl₂ on the clotting time in blood of differentconcentrations of lyophilized Thr and Mag˜Thr.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention involves the synthesis of various nanoparticles,the introduction of reactive chemical groups onto the surface of saidnanoparticles, wherein said reactive chemical groups are most preferablyaldehyde groups and carbon-carbon activated double bonds, and thesubsequent attachment of thrombin molecules onto said modified surface,either covalently or physically, as will be described in more detailbelow.

According to one preferred embodiment of the invention, thenanoparticles comprise iron oxide. Magnetic iron oxide-containingnanoparticles (hereinafter sometimes desiganted Mag nanoparticles orMag) may be prepared following the synthetic procedures described byMargel et al. in international publication no. WO 99/62079, incorporatedherein by reference, according to which a suitable polymeric chelatingagent, such as gelatin, is contacted in aqueous solution with a sourceof iron ions under well-defined conditions, to yield Mag nanoparticleshaving diameters ranging from 15 to 100 nm. The introduction of reactivechemical groups onto the surface of the Mag nanoparticles may beaccomplished by the methods described in WO 99/62079, according to whichsaid nanoparticles are treated with dextran or gelatine to form afunctional coating thereon, which contains suitable reactive groups thatmay be used for the subsequent attachment of thrombin. Alternatively,following the formation of the dextran coating, a suspension of thecoated Mag nanoparticles is reacted with suitable reagents, capable offorming new chemically reactive groups on said dextran coating.Particularly preferred reagents are oxidizing agents or divinyl sulfone(DVS) or derivatives thereof. Preferably, the dextran-coated Magnanoparticles are reacted with divinyl sulfone at elevated temperature,under alkaline pH, whereby reactive carbon-carbon double bonds areobtained on said nanoparticles.

According to another preferred embodiment of the invention, thenanoparticles are made of albumin (hereinafter sometimes designated ABnanoparticles or AB). Albumin nanoparticles may be prepared by adding anappropriate organic solvent into an aqueous solution of human serumalbumin at room temperature, whereby the albumin is caused toprecipitate. Thereafter, the temperature is preferably raised, in orderto harden the albumin particles. The size of the AB nanoparticles may becontrolled by changing experimental parameters such as albuminconcentration or organic solvent type. Typical concentration of thealbumin in the aqueous solution is in the range of 2%-4% (w/w).Preferred organic solvents that may be used for the precipitation ofalbumin are lower alkanols, such as ethanol. The introduction ofreactive chemical groups onto the surface of the AB nanoparticles ismost preferably accomplished by reacting said AB nanoparticles withglutaraldehyde (GA) or DVS and derivatives thereof, to obtain aldehydereactive groups or carbon-carbon double bonds on said surface,respectively.

According to another preferred embodiment of the invention, thenanoparticles comprise silica. Hereinafter, these silica containingnanoparticles are sometimes designated SiO₂ nanoparticles or SiO₂.

In one preferred variant, the SiO₂ nanoparticles are provided in theform of an organic polymeric core coated with one or more silica shells,optionally having a magnetic layer interposed between said core and saidsilica shell(s). Preferably, the organic core comprises a polymerselected from the group consisting of polystyrene,polymethylmetacrylate, polychloromethylstyerne and polyvinyltoluene.Suitable synthetic routes for the preparation of said nanoparticles aredescribed by H. Bamnolker and S. Margel [J. of Polymer Science, PolymerChemistry 34, 1857 (1996) and U.S. Pat. No. 6,103,379] and S. Brandrissand S. Margel [Langmuir 9, 1232 (1993)], which are incorporated hereinby reference. A preferred procedure involves the dispersionpolymerization of a suitable monomer, such as styrene, in ethanol in thepresence of polyvinylpyrrolidone as surfactant, to give monodispersedpolystyrene microspheres of diameter ranging between 0.3-6 μm, followingwhich silica nanoparticles of substantially smaller size (ca. 30 nm) arecoated on the polystyrene particles by seeded polymerization oftetraethoxysilane on the surface of said polystyrene particles, usingthe Stober method. If desired, an intermediate magnetic layer may beinterposed between the organic core and the silica coating by means ofseeded polymerization of iron salts on said core according to aprocedure similar to that described by Margel et al. [Scientific andClinical Applications of Magnetic Carriers, Ed. U. Hafeli, W. Schutt, J.Teller and M. Zborowski, Plenum Press, N.Y. 37-51 (1997)]. Optionally,hollow silica nanoparticles may be obtained by burning the organic coreaccording to the methods described in U.S. Pat. No. 6,103,379.

In a second preferred variant, silica nanoparticles having diameters inthe range of 10-100 nm are provided. These nanoparticles areconveniently prepared by polymerization of Si(OEt)₄ according to theStober Method (see, for example, Stober et al., J. of. Colloid andInterface Science, 26, p. 62 (1968) and Brandriss and Margel, Langmuir9, 1232 (1993), which are incorporated herein by reference). SiO₂nanoparticles of different sizes within the range of 10-100 nm areprepared by changing the Stober polymerization conditions, i.e. themonomer [(Si(OEt)₄] concentration, the water concentration, ammonia,etc, as described in detail in the aforementioned publications

The introduction of reactive chemical groups onto the surface of thesilica nanoparticles (either the silica nanoparticles that are providedin the form of organic polymeric core coated with one or more silicashells, optionally having magnetic layer interposed between said coreand said silica shell(s), or the silica nanoparticles having diametersin the range of 10-100 nm) may be carried out according to theprocedures described by Brandriss and Margel [Langmuir 9, 1232 (1993)].According to one preferred procedure, the silica nanoparticles are mixedtogether with a reagent of the formula Si(O-Alk)₃(CH₂)nNH₂, wherein Alkis a lower alkyl group, which is most preferably methyl or ethyl, and nis an integer varying from 2 to 10, in a suitable organic solvent, whichis most preferably a lower (C₁-C₄) alkanol, at a temperature in therange of 20 to 50° C. for several hours, following which thenanoparticles are washed to remove excess of reagents therefrom.Alternatively, the reaction between the silica-containing nanoparticlesand the reagent of the formula Si(O-Alk)₃(CH₂)nNH₂ as defined above iscarried out in aqueous solution under suitable conditions, and mostpreferably in acetate buffer at pH 5.5, according to the description ofWiksteron et al. [J. of Chromatography 455, p. 105 (1988)]. Theresulting silica nanoparticles have primary amino groups on theirsurface, which are subsequently reacted in water or in a suitableorganic solvent with a reagent such as acryloyl chloride, DVS orderivatives thereof to introduce an activated carbon-carbon double bondonto the surface of said silica nanoparticles.

The attachment of thrombin molecules to the nanoparticles havingreactive chemicals groups on their surface may be accomplishedchemically, by allowing said thrombin molecules to form covalent bondseither directly with said reactive groups, or with a suitable spacer armthat is bonded to said reactive groups.

According to one preferred embodiment of the invention, the directcovalent binding of thrombin to the nanoparticles is most preferablycarried out by allowing the primary amine groups of the thrombinmolecules to react either with the activated double bonds provided onthe surface of the nanoparticles via a Michael addition reaction, orwith the aldehyde groups provided on said surface through the formationof Schiff Bases. In general, preferred reaction conditions includesuspending the nanoparticles having either activated double bonds oraldehyde groups on their surface in a suitable solvent, which is mostpreferably water, under alkaline pH, and adding thereto thrombinmolecules such that the weight ratio between said nanoparticles and saidthrombin molecules is preferably in the range of 100:1 to 5:1, followingwhich the reaction mixture is shaken at room temperature for a period oftime ranging from 30 minutes to several hours. Subsequently, residualactivated double bonds or aldehyde groups are blocked using appropriatereagents, such as glycine, ethanolamine or other amino containingcompounds, and the thrombin-conjugated nanoparticles are isolated fromthe reaction mixture using conventional separation techniques, such ascentrifugation or magnetic separation methods, if applicable.Hereinafter, thrombin conjugated nanoparticles having thrombin moleculesdirectly covalently bonded to reactive groups provided on the surface ofthe nanoparticles are designated X-Thr, wherein X identifies thenanoparticles (e.g., X is Mag, AB or SiO₂) and Thr indicates thrombin.

According to another preferred embodiment of the invention, a suitablespacer arm is covalently linked to the reactive groups provided on thesurface of the nanoparticles, following which the thrombin molecules arecaused to form chemical bonds with said spacer arm. A suitable spacerarm is most preferably a molecule having at least two functional groups,which may be identical or different, wherein the first functional groupis capable of forming a covalent bond with the reactive chemical groupsprovided on the surface of the nanoparticles, and the second functionalgroup is capable of subsequently forming a covalent bond with thethrombin molecules.

A particularly preferred molecule that may be used as a spacer armaccording to the present invention has a first functional group which isa primary amine group, that may either undergo a Michael addition orform a Schiff base with the activated double bonds or the aldehydegroups provided on the nanoparticles, respectively, under the conditionsdescribed hereinabove for the reactions between the variousnanoparticles and thrombin, and a second functional group capable ofparticipating in a subsequent chemical reaction with reactive groups ofthe thrombin molecules. Preferably, the second functional group of thespacer arm is a carboxylate group, such that the covalent binding of thethrombin to the spacer arm may be accomplished via the carboiimideactivation method, according to the reaction conditions that are asdescribed by Melamed and Margel [J. of Colloid and Interface Science241, 357 (2001)], which is incorporated herein by reference.Alternatively, the spacer arm may have a second functional group that isselected from among primary amine, aldehyde, carbon-carbon double bondsand hydroxyl. It may be readily appreciated that in the case wherein thefirst and second functional groups of the spacer arm are identical (forexample, both groups are primary amine groups), then an excess of saidspacer arm should be used in the reaction with the nanoparticles.

Preferred molecules that may be used as spacer arms are selected fromthe group consisting of BSA (bovine serum albumine), glutaraldehyde and1-amino hexanoic acid. Hereinafter, thrombin conjugated nanoparticleswherein a spacer arm is covalently linked to reactive groups provided onthe surface of the nanoparticles, with the thrombin molecules formingcovalent bonds with said spacer arm are sometimes designatedX-Spacer-Thr, wherein X identifies the nanoparticle (e.g., Mag, AB orSiO₂), Spacer identifies the molecule used as a spacer arm (e.g., BSA)and Thr indicates thrombin.

In another aspect, the present invention encompasses thrombin conjugatednanoparticles, wherein the thrombin molecules are physically adsorbedonto spacer arms, and wherein said spacer arms are covalently-linked tothe surface of the nanoparticles. The attachment of the thrombinmolecules to the nanoparticles is accomplished by causing said thrombinmolecules to become physically adsorbed onto a suitable spacer arm thatis bonded to the reactive groups provided on the surface of thenanoparticles. A spacer arm which is capable of strongly attractingthrombin through physical adsorption is most preferably albumin.Typically, following the introduction of the spacer arm onto the surfaceof the nanoparticles utilizing the synthetic procedures described above,the nanoparticles having the spacer arm covalently linked thereto aresuspended in an alkaline aqueous solution, the pH of said solution beingin the range of 7 to 9, at a temperature not higher than 37° C.,following which thrombin is added to the suspension, such that theweight ratio thrombin:nanoparticles is preferably in the range of 1:100to 1:5, to obtain the desired thrombin-conjugated nanoparticles.Hereinafter, thrombin conjugated nanoparticles that are based on thephysical binding of thrombin to the nanoparticles through a spacer armare sometimes labeled as follows: X˜Thr, wherein X identifies thenanoparticle (e.g., Mag, AB, SiO₂, etc).

The thrombin-conjugated nanoparticles according to the invention haveuseful therapeutic properties in the treatment and/or prophylaxis ofconditions associated with the blood clotting process.

More specifically, the thrombin-conjugated nanoparticles according tothe present invention allow rapid and effective interaction of thrombinwith fibrinogen in aqueous solution, plasma and blood, to form thedesired fibrin clot, said clot being characterized by the presence ofnanoparticles entrapped within its polymeric network.

It has been found that the thrombin conjugated to the nanoparticlesaccording to the present invention possesses different properties fromthose of the free enzyme, i.e. different clotting time, andsignificantly increased stability towards various potentially disruptiveconditions, e.g. inhibitors, bacteria, increased temperature, storagetime, pH, light, lyophilization, etc. The fibrin sealant formed with thethrombin-conjugated nanoparticles of the present invention is also saferthan the sealant formed with free thrombin, particularly for allergicpatients, since significant leaching of free thrombin from theconjugated nanoparticles is prevented. Under similar thrombinconcentrations, the clotting time with fibrinogen of thephysically-adsorbed thrombin nanoparticles was shorter than that of thecovalently bonded thrombin nanoparticles. Also, under similar thrombinconcentrations, the clotting time of the thrombin conjugatednanoparticles was according to the following order: blood<plasma<aqueoussolution. An opposite behavior was observed for the free enzyme, i.e.plasma<blood. A significant acceleration in the clotting time of thethrombin-conjugated nanoparticles was observed by adding CaCl₂ to theconjugated nanoparticles-containing aqueous suspension. A similaracceleration effect of CaCl₂ was observed for the free enzyme, butsignificantly more moderate. A moderate acceleration in the clottingtime of both the free and immobilized thrombin was also observed byadding factor XIII to the thrombin or thrombin-conjugated nanoparticlescontaining aqueous suspension.

Accordingly, the present invention provides the thrombin-conjugatednanoparticles for use as a therapeutic substance. More particularly, thepresent invention provides the thrombin-conjugated nanoparticles for usein the treatment of conditions associated with blood clotting process.

In a further aspect, the present invention provides a therapeuticcomposition comprising thrombin-conjugated nanoparticles together with asuitable carrier for treating conditions associated with blood clottingprocess. It should be noted that the therapeutic composition accordingto the present invention may be either in a solid form or in a liquidform.

According to one preferred embodiment, the thrombin-conjugatednanoparticles will be provided in a therapeutic composition in the formof a solid, dry powder that is suitable for topical administration tothe desired body site. It has been found that a particularly stablecomposition comprising dried thrombin-conjugated nanoparticles may beobtained by lyophilization of an aqueous suspension of thethrombin-conjugated nanoparticles in the presence of one or moresuitable dispersants or surfactants, such as gelatin orpolyvinylpyrrolidone, gelatin being most preferred. According to aparticularly preferred embodiment, the weight ratio between thethrombin-conjugated nanoparticles and the gelatin in the aqueoussuspension before the lyophilization is in the range of 1:2 to 2:1.Accordingly, a solid therapeutic composition comprising driedthrombin-conjugated nanoparticles and at least one dispersant, which ismost preferably gelatin, forms another aspect of the present invention.The dried thrombin-conjugated nanoparticles can be stored at 4° C. or atroom temperature for at least 6 months without significant loss of theirclotting time with fibrinogen.

In another preferred embodiment of the present invention, thethrombin-conjugated nanoparticles are provided in the form of a soliddry powder supported on a suitable carrier such as cellulose, collagenor gelatin sheets. For example, covering a sheet of collagen with thethrombin-conjugated aqueous suspension and subsequently lyophilizing thesame will give the desired supported nanoparticles.

According to another preferred embodiment, the thrombin-conjugatednanoparticles will be provided in a therapeutic composition that is in aliquid form. To this end, thrombin-conjugated nanoparticles of thepresent invention per se, or thrombin-conjugated nanoparticles obtainedfollowing lyophilization in the presence of gelatin, as described above,may be re-suspended in a suitable liquid vehicle, which is mostpreferably water, in a weight concentration ranging between 0.01 to 5%.The diameter of the dispersed thrombin conjugated nanoparticles issimilar to the diameter of the nanoparticles before lyophilization.

It should be noted that suitable additives, such as Ca salts (e.g.,CaCl₂), factor XIII and antifibrinolytic agents such as aprotinin ortranexamic acid may be present in the aforementioned therapeuticcompositions of the present invention. These additives may be introducedto the aqueous suspension containing the thrombin-conjugatednanoparticles and (optionally) the dispersant prior to thelyophilization.

The thrombin-conjugated nanoparticles of the present invention may beused, or adapted for use, in connection with a very broad range ofclinical conditions and situations. Thus, the thrombin-conjugatednanoparticles disclosed and claimed herein may be used to assist normalhemostasis, induce hemostasis in hemophilia patients and to causeaccelerated wound healing. In addition, they may be employed incircumstances requiring the use of a tissue glue (for example, in theanchoring of medical devices to living tissue). Finally, thethrombin-conjugated nanoparticles may be used in many, if not all, areasof surgical practice, including general surgery, orthopedics, urology,plastic surgery, gynecology, dental surgery, circumcision andcardiovascular surgery. The stability of the presently-claimednanoparticles, together with their relative simplicity of use, rendersthem suitable for use in the aforementioned clinical indications in awide range of different settings including in the armed services (e.g.in field hospitals and on the battlefield), home care, inpatient andoutpatient clinics and operating suites, as well as in variousindustrial settings.

Accordingly, the present invention provides a method for preparingfibrin-containing biological sealant, wherein said method comprisescontacting fibrinogen with a therapeutically effective amount ofthrombin-conjugated nanoparticles of the present invention. Preferably,the fibrinogen is contacted with a dry powder comprisingthrombin-conjugated nanoparticles and a dispersant, wherein saiddispersant is most preferably gelatin, in a liquid medium selected fromthe group consisting of aqueous solution, plasma or blood, whereby thefibrin sealant is formed. Most preferably, the thrombin-conjugatednanoparticles and fibrinogen are contacted in the presence of calciumions or factor XIII.

The presently-claimed thrombin-conjugated nanoparticles are intended foruse in the context of enhanced blood clotting and wound closure andrepair, all of which are surface phenomena. Consequently, thetherapeutically-effective amount of the thrombin-conjugatednanoparticles to be used in the above-defined method, and in thepreparation of the above-defined therapeutical composition, is to beunderstood as an amount appropriate for the surface area of the wound tobe treated. In practice, this means that the user will select an amountof the nanoparticle preparation that is sufficient to either partiallycover or fully cover the wound area.

It should be noted that the thrombin-conjugated nanoparticles of thepresent invention may also be used for additional purposes, e.g.controlled release by encapsulation or surface binding of an appropriatedrug (e.g. antibiotics) or for targeting by binding to the surface inaddition to thrombin an homing reagent. To this end, thrombin was boundcovalently or physically to nanoparticles containing encapsulatedantibiotics, e.g. Ampicillin, by using similar procedures to thatdescribed above, substituting the nanoparticles for nanoparticlescontaining the encapsulated Ampicillin.

Homing agents, e.g. Ampicillin, for targeting was bound to thenanoparticles by using similar procedures to that described above,substituting the blocking step of residual activating groups (aftercompleting the thrombin binding) with glycine for the binding of thehoming agent.

EXAMPLES

1. Materials and Methods

1-A. Materials

All reagents used in this work were of analytical grade from commercialsources. Thrombin (from bovine plasma, 50 U/mg)) was purchased fromMerck; Antithrombin III (from bovine plasma), Fibrinogen (fraction I,type I-S from bovine plasma), Gelatin 60 and 300 from porcine skin,Human serum albumin (HSA), Bovine serum albumin (BSA) and Ampicillinsodium salt were purchased from Sigma; Factor XIII (Fibrogammin P, 3.4U/mg) was purchased from Aventis Behring GmbH, Germany;D-Phenylalanyl-L-pipecolyl-L-arginine-p-nitroanilide dihydrochloride(S-2238, a substrate for thrombin) was purchased from Chromogenix,Sweden; Plasma (human normal control plasma) was purchased fromDiagnostica STAGO, France. Water was purified by passing deionized waterthrough an Elgastat Spectrum reverse osmosis system (Elga Ltd., HighWycombe, UK).

1-B. Diameter and Size Distribution

The diameter and size distribution of the various nanoparticles weredetermined by scanning electron microscopy and transmission electronmicroscopy (JSM-840, Jeol), and sub-micron and micron size particleanalyzers of Coulters Electronics, UK.

1-C. Blood Clotting Measurements

Blood clotting measurements in fibrinogen solution, plasma and blood ofthrombin and thrombin immobilized to the various nanoparticles wereperformed with the STart-4 equipment supplied by Diagnostica Stago,France. The reported clotting time values are an average of at least 4measurements of each type of Thr-immobilized nanoparticles.

1-D. Sterilization of the Thrombin Mag Conjugated Nanoparticles

Sterilization of the Mag nanoparticles coated with dextran and activatedwith divinyl sulfone was performed by means of the following two steps:(1) passing the nanoparticle aqueous suspension through 0.2 μm filterpaper; (2) sterilizing the nanoparticle suspension with a standardautoclave at high pressure and 120° C. for 20 min. The magnetic columnsas well as the other necessary tools were also sterilized byautoclaving. The binding of the BSA was then performed in a laminar hoodunder sterile conditions. The sterilized nanoparticle suspension (in0.1M PBS) was then dispensed (in 100 μl aliquots) into small vials.These vials were then either stored at 4° C. or their contentslyophilized, until required for use.

1-E. Measuring the Amount of Bound Thrombin

The amount of bound thrombin was determined with the thrombin substratechromogenix reagent S-2238. Briefly, 1.9 ml of 0.1M Tris buffer at pH8.3 were added to 0.2 ml aqueous suspension of the thrombin-conjugatednanoparticles. Then, 0.1 ml of the reagent S-2238 were added. The tubewas then shaken for 2 min at room temperature. Thereafter, thesupernatant of the nanoparticle suspension was separated from thenanoparticles themselves by centrifugation, ultrafiltration or magneticseparation. The absorption of the supernatant at 405 nm was thenmeasured. The amount of thrombin conjugated to the various nanoparticleswas determined according to the supernatant absorption intensity at 405nm, according to a calibration curve of known concentrations of freethrombin.

Preparation 1 Synthesis of Iron Oxide-Containing Nanoparticles andSurface Modification Thereof

Synthesis

10 ml of deairated aqueous solution containing 10 mg gelatin (300 bloom)and 0.07 mmol FeCl₂.4H₂O was shaken at 60° C. for 10 min. 0.014 mmolNaNO₂ was added to the solution, and after 10 min the pH of the solutionwas increased to 9.5 by the addition of 0.3M NaOH solution. The solutionwas shaken for another 40 min. Thereafter, the procedure of thesuccessive addition of FeCl₂.4H₂O NaNO₂ and NaOH to pH 9.5 was repeatedthree times. The formed magnetic nanoparticles were then cleaned fromexcess reagents by the use of magnetic columns. Transmission ElectronMicroscopy studies and coulter measurements demonstrated that theaverage diameter of the dried Mag nanoparticles is 20 nm±10% and thehydrodynamic average diameter of these Mag nanoparticles dispersed inwater is 70 nm±10%.

Mag nanoparticles containing encapsulated antibiotics such as Ampicillinsodium salt were prepared by using a similar procedure, in which theaforementioned 10 ml aqueous solution containing 10 mg gelatin wassubstituted by a 10 ml aqueous solution containing 2 mg Ampicillinsodium salt and 10 mg gelatin.

Surface Modification

The Mag nanoparticles (20 nm average dry diameter) obtained according tothe procedure described above were shaken in water at 85° C. in thepresence of 2% (w/v) dextran (MW 37,5000) for 1 h, to form a suspension,which was allowed to cool to room temperature. The dextran-coatednanoparticles were then washed from excess dextran by magnetic columns.0.66 ml DVS was then added into a 40 ml aqueous suspension containing100 mg of the washed dextran coated Mag nanoparticles at 60° C.Thereafter, triethyl amine was added gradually to the nanoparticlesuspension, until the pH reached pH 10.5. The suspension was then shakenat 60° C. for approximately 12 h. Excess DVS was then washed from themodified nanoparticles by means of magnetic columns. The modified Magnanoparticle aqueous suspension was then stored at 4° C.

Modification of the surface of the Ampicillin encapsulated Magnanoparticles was performed similarly, substituting the Magnanoparticles with Ampicillin encapsulated Mag nanoparticles.

Preparation 2 Synthesis of Albumin Nanoparticles and SurfaceModification Thereof

Synthesis

An aqueous solution (25 ml) containing 1 g human serum albumin wasshaken at room temperature for 15 minutes, following which 50 ml ofn-propanol were added thereto, to obtain particles of 0.7-1.5 μmdiameter. The temperature was then raised to 50° C. for 15 min, and thento 82° C. (the boiling point of n-propanol) for 1 h. The ABnanoparticles were then washed extensively by several centrifugationcycles in aqueous solution.

AB nanoparticles containing encapsulated antibiotics such as Ampicillinsodium salt were prepared by using a procedure similar to that describedabove, substituting the 25 ml aqueous solution containing 1 g humanserum albumin with a 25 ml aqueous solution containing 2 mg Ampicillinsodium salt and 1 g human serum albumin.

Surface Modification

4 ml of glutaraldehyde 25% were added to 100 ml of pure water containing1 g of dispersed AB particles obtained according to the proceduredescribed above. The suspension was then shaken at room temperature for3 h. The resulting nanoparticles were then washed extensively fromexcess glutaraldehyde by several centrifugation cycles in pure water,and then stored at 4° C.

Modification of the Ampicillin encapsulated AB nanoparticles wasperformed similarly, by substituting the AB nanoparticles withAmpicillin encapsulated AB nanoparticles.

Preparation 3 Synthesis of Silica Nanoparticles Deposited on an Organic(Polystyrene) Core and Surface Modification Thereof

Synthesis

Polystyrene particles of 2.3 μm±10% (1 g) were added to a flaskcontaining ethanol (93.6 ml) and distilled water (1.9 ml), and themixture was sonicated to disperse the particles. Ammonium hydroxide (1.3ml) and Si(OEt)₄ (3.2 ml) were then added, and the suspension was shakenat room temperature for 12 h. The resulting SiO₂ coated polystyrenenanoparticles were freed from free silica nanoparticles (ca. 30 nmdiameter) by repeated centrifugation cycles. The SiO₂ coated polystyrenenanoparticles were then dried in a vacuum oven. The content of thesilica coating on the polystyrene particles was increased by repeatingthe aforesaid coating procedure. The silica content of the core-shellparticles was 7.8% and 13.8% (w/w) after the first and second coatingcycles, respectively. Scanning electron micrographs and cross sectionaltransmission electron micrographs demonstrated the complete coverage ofthe polystyrene core particles with a few layers of silica nanoparticlesof approximately 30 nm average diameter.

Surface Modification

Dry SiO₂ coated polystyrene nanoparticles having an average size of 2.3μl (100 mg) were introduced into a triple-neck flask containing 80 ml0.1 M acetate buffer, pH 5.5. The mixture was then sonicated in order todisperse the particles, following which 0.4 ml of the amphiphileSi(OEt)₃(CH₂)₃NH₂ was added to the suspended particles. The suspensionwas then mechanically stirred at 60° C. for 12 h. Thereafter, thederivatized particles were washed by a few centrifugation cycles inacetate buffer, and then in distilled water. The resulting SiO₂ coatedpolystyrene nanoparticles, bearing amino groups on their surface weredispersed in water and stored at 4° C. prior to use.

The aforementioned SiO₂ coated polystyrene nanoparticles, having aminogroups provided on their surface (300 mg) were introduced into a flaskcontaining 10 ml bicarbonate buffer, 0.1 M at pH 8.3. The mixture wasthen sonicated in order to disperse the particles. 0.1 ml acryloylchloride was then added to the suspension. The suspension was shaken atroom temperature for 1 h. Excess acryloyl chloride was removed by a fewcentrifugation cycles in water. The resulting particles (SiO₂ coatedpolystyrene nanoparticles having activated double bonds on theirsurface) were dispersed in water and then stored at 4° C. prior to use.

Preparation 4 Synthesis of Silica Nanoparticles Deposited on an Organic(Polystyrene) Core, Having a Magnetic Layer Interposed Therebetween, andSurface Modification Thereof

Polystyrene nanoparticles of average size 2.3 μm (1 g) were added to aflask containing 20 ml distilled water, following which the mixture wassonicated for a few minutes and then mechanically stirred at ca. 200rpm. The temperature was then preset to 60° C. Nitrogen was bubbledthrough the suspension during the coating process to exclude air. 0.1 mlof iron chloride tetrahydrate aqueous solution (1.2 mmol in 10 ml H₂O)and 0.1 ml of sodium nitrite aqueous solution (0.02 mmol in 10 ml H₂O)were then successively introduced into the reaction flask. Thereafter, asodium hydroxide aqueous solution (0.5 mmol in 10 ml H₂O) was addeduntil a pH of ca. 10 was reached. This procedure was repeated fourtimes. During this coating process the core polystyrene nanoparticlesbecame brown-black colored. The suspension was then cooled to roomtemperature. The formed magnetic polystyrene particles were then washedextensively in water with a magnet and dried in a vacuum oven. Themagnetic polystyrene particles were coated with silica nanoparticles(ca. 30 nm), according to the procedure described in preparation 3.

Surface Modification

The introduction of reactive chemical groups onto the surface of themagnetic SiO₂ coated polystyrene nanoparticles was accomplishedsimilarly to the procedure of preparation 3.

Preparation 5 Synthesis of Silica Nanoparticles of 10-100 nm Diameterand Surface Modification Thereof

Synthesis

The following reagents were successively added into a flask: ethanol(93.6 ml), distilled water (1.5 ml), ammonium hydroxide (1.3 ml) andSi(OEt)₄ (2.8 ml). The resulting solution was then shaken at roomtemperature for ca. 5 h. The formed nanoparticles were washed byevaporation of the unreacted monomer, ethanol and ammonia. Water wasadded during the evaporation to retain the total volume of the silicasuspension. The particles obtained were of ca. 10 nm±15% diameter.

Surface Modification

The introduction of reactive chemical groups onto the surface of thesilica nanoparticles having diameter of approximately 10 nm wasaccomplished similarly to the procedure of preparation 3, with thefollowing changes:

At the stage of introducing the primary amine groups, the buffer acetateat pH 5.5 was replaced with buffered water at pH 9.5, and the subsequentcentrifugation was substituted with dialysis against water.

At the stage of introducing the double bond groups, centrifugation wassubstituted with dialysis against water.

Preparation 6 Covalent Binding of a Spacer Arm (Bovine Serum Albumin(BSA)) to Nanoparticles having Activated Double Bonds or Aldehyde Groupson their Surface

BSA (10 mg) was added to vials containing 100 mg nanoparticles havingeither activated double bonds or aldehyde groups on their surface(obtained according to any of the preparations described above),suspended in 40 ml 0.1M bicarbonate buffer at pH 8.3. The suspension wasshaken at room temperature for ca. 12 hours. Blocking of residualchemically reactive groups (activated double bonds or aldehydes) of theBSA conjugated particles was then performed by adding 400 mg glycine toeach vial. The particle suspension was then shaken at room temperaturefor additional 2 hours, following which the BSA-conjugated nanoparticleswere separated from excess BSA and glycine by centrifugation cycles ormagnetic separation with 0.1M PBS (pH 7.4). The BSA-conjugatednanoparticles were then stored at 4° C. up. The amount of BSA bound tothe various nanoparticles was measured according to Lowry method [J.Biol. Chem. 1951, 193, 265.]

Preparation 7 Direct Chemical Binding of Thrombin to NanoparticlesHaving Activated Double Bonds or Aldehyde Groups on Their Surface

The direct covalent binding of thrombin to nanoparticles having eitheractivated double bonds or aldehyde groups on their surface (obtainedaccording to preparations 1 to 5 described above) was accomplishedsimilarly to the procedure of preparation 6, by replacing BSA withthrombin.

Preparation 8 Chemical Binding of Thrombin to Nanoparticles Through aBSA Spacer

123 mg NHS (Acetic acid N-hydroxysuccinimide ester) and 82 mg CMC(1-Cyclohexyl-3-(2-morpholinoethyl) carbodiimidemetho-p-toluenesulfonate) were added to a suspension containing 100 mgnanoparticles conjugated with BSA (prepared according to preparation 6)suspended in 40 ml 0.1M phosphate buffer at pH 6.5. The nanoparticlessuspension was then shaken at room temperature for 4 h. The activatedconjugated nanoparticles were then separated from excess activatingreagents by centrifugation cycles or magnetic separation with 0.1M PBS(pH—7.4). 10 mg thrombin was then added to the washed suspension,following which the suspension was shaken at room temperature for ca. 12h. Blocking of residual activating groups was accomplished by adding 400mg glycine to the nanoparticle suspension, and then shaking thesuspension at room temperature for an additional 3 h. Thethrombin-conjugated nanoparticles were then washed from unbound thrombinand glycine by means of either several cycles of centrifugation ormagnetic separation with 0.1M PBS (pH—7.4). The thrombin-conjugatednanoparticles were then lyophilized or stored at 4° C. prior to use. Theamount of thrombin conjugated to the nanoparticles was determined withthe chromogenix reagent S-2238, according to the description givenhereinabove.

Preparation 9 Physical Binding of Thrombin to Nanoparticles Having BSASpacers

5 mg thrombin were added to a 0.1M PBS (pH 7.4) suspension containing100 mg BSA-conjugated nanoparticles (that were prepared according topreparation 6 above). The suspension was then shaken at room temperaturefor ca. 12 h. The thrombin-conjugated nanoparticles were then washedfrom unbound thrombin by centrifugation cycles or magnetic separationwith 0.1M PBS (pH—7.4). The thrombin-conjugated nanoparticles were thenlyophilized or stored at 4° C. prior to use. The amount ofthrombin-conjugated to the nanoparticles was determined with thechromogenix reagent S-2238, according to the description givenhereinabove.

Example 1 Extent of Leakage of Thrombin (Thr) Immobilized to VariousNanoparticles Suspended in PBS in the Absence or Presence of 4% HumanSerum Albumin (HSA)

Vials containing nanoparticles bonded with 37 μg Thr suspended in 120 μl0.1 M PBS (pH=7.4) in the absence or presence of 4% HSA were shaken atroom temperature. At various time intervals the Thr immobilizedparticles were removed from the supernatants. The amount of leached Thrin the supernatants was then measured as described in the foregoingexperimental section. The results are indicated in Table 1. TABLE 1Extent of leakage of Thr immobilized to various nanoparticles suspendedin PBS in the absence (A) or presence (B) of 4% HSA. Leached Thr(%)Thrombin-conjugated Time (h) nanoparticle 0.30 4 24 A Mag˜Thr 0 0.070.13 AB˜Thr 0 0 0 SiO₂˜Thr (2.3 μm) 1.9 6.4 11.2 B Mag˜Thr 0 1.9 6.5AB˜Thr 0 0 0 SiO₂˜Thr (2.3 μm) 2.4 11.4 16.5

The results shown in Table 1 indicate that there was no leakage of Thrto PBS, or PBS containing 4% HSA, from Thr immobilized to ABnanoparticles suspended in PBS. Table 1 also shows that the extent ofleakage of Thr from Thr immobilized to Mag nanoparticles suspended inPBS is very small, i.e. after 0.5 h no leakage was detected; after 4 h,0.07% and 1.9% of Thr was leached to PBS and PBS containing 4% HSA,respectively. The highest degree of leakage of Thr was observed forSiO₂˜Thr, i.e. after 0.5 h 1.9% and 2.4% of Thr was leached to PBS andPBS containing 4% HSA, respectively. It should however be noted thatusually the realistic clotting time in plasma, or blood, of the Thrimmobilized nanoparticles is less than 1 min, and in that period of timethe extent of leached Thr to PBS containing 4% HSA was negligible (lessthan 0.3%). It should also be noted that for the covalent Thr-bondednanoparticles no leakage was detected even after 24 h.

In all the experiments described above the clotting times in plasma ofthe supernatants removed from the nanoparticles were also tested.However, plasma clotting was not observed up to 500 seconds, at whichtime the experiments were terminated.

Example 2 Effect of Diameter of the SiO₂ Nanoparticles on the ClottingTime in Plasma of SiO₂˜Thr Suspended in PBS

The clotting time was measured at 37° C. by mixing 50 μl plasma withsamples containing 32 μg of Thr immobilized to SiO₂ nanoparticles ofvarious diameters suspended in 100 μl 0.1M PBS (pH=7.4). The results areshown in the following table. TABLE 2 Effect of the diameter of the SiO₂nanoparticles on the clotting time in plasma of SiO₂˜Thr suspended inPBS. Diameter Particles Bound Thr Clotting time (μm) (mg) (μg) (sec)0.01 0.4 32 15.2 ± 0.3 0.75 3.9 32 32.3 ± 3.6 2.3 9.1 32 25.4 ± 2.5 311.4 32 23.6 ± 2.6 4.5 16.8 32 37.8 ± 2.7

Example 3 Effect of the inhibitor Antithrombin III on the activity ofThr and immobilized Thr: SiO₂˜Thr (2.3 μm) (A), AB˜Thr (B) and Mag˜Thr(C) Suspended in PBS

Vials containing 40 μg Thr or Thr bonded nanoparticles suspended in 200μl 0.1 M PBS (pH=7.4) in the presence of different concentrations ofAntithrombin III were shaken at RT for 1 h. The Thr immobilizedparticles were then removed from the supernatant. The remained activityof Thr and Thr bonded nanoparticles was then measured as described inthe experimental part. FIG. 1 demonstrates the significant stabilizationof Thr against inhibitors (e.g. antithrombin III) obtained byimmobilizing the Thr to the various nanoparticles (SiO₂, AB and Mag).For example, in the presence of 5 units of antithrombin III, the freeThr lost about 75% of its original activity while Thr immobilized to Maglost about 2% of its original activity.

Example 4 Effect of 8.3 mM CaCl₂ on the Clotting Time in Plasma andBlood of Thr and SiO₂˜Thr (2.3 μm), AB˜Thr and Mag˜Thr ˜Suspended in PBS

Clotting time was measured at 37° C. by mixing 60 μl plasma (or blood)with samples containing, each sample, 13 μg of Thr or Thr immobilized tovarious nanoparticles suspended in 100 μl 0.1M PBS (pH=7.4) in theabsence or presence of 8.3 mM CaCl₂. The results are shown table 3.TABLE 3 Effect of 8.3 mM CaCl₂ on the clotting time in plasma and bloodof Thr and SiO2˜Thr (2.3 μm), AB˜Thr and Mag˜Thr suspended in PBS.Thrombin Coagulation time Coagulation time conjugated CaCl₂ in plasma inblood nanoparticle (mM) (sec) (sec) Mag˜Thr 0 47.4 ± 1.3 33.1 ± 1.3Mag˜Thr 8.3 19.7 ± 1.1 14.2 ± 1.3 AB˜Thr 0 39.8 ± 2.7 27.8 ± 3.5 AB˜Thr8.3 17.5 ± 1.1 13.7 ± 3.2 SiO₂˜Thr 0 60.7 ± 3.5 44.3 ± 2.3 SiO₂˜Thr 8.331.1 ± 2.2 25.2 ± 2.2 Thr 0 21.5 ± 1.8 25.3 ± 2.1 Thr 8.3 12.3 ± 2.516.7 ± 2.5Table 3 illustrates the following:(1). The clotting time of Thr in plasma is shorter than in blood.Contrarily, the clotting time of the immobilized Thr nanoparticles inplasma is longer than in blood.(2). Addition of 8.3 mM CaCl₂ to plasma or blood leads to decrease inthe clotting time of both Thr and the immobilized Thr nanoparticles.However, this effect is significantly more prominent for the immobilizedThr nanoparticles than for the Thr.3). Under the described experimental conditions, the clotting times inblood containing additional 8.3 mM CaCl₂ and AB˜Thr and Mag˜Thr areshorter than that of Thr, i.e. 13.7, 14.2 and 16.7 sec., respectively.

Example 5 Clotting Time in Plasma as Function of Storage at RT of Thrand SiO₂˜Thr Suspended in PBS

Clotting time was measured at 37° C. by mixing 50 μl plasma with samplescontaining, each sample, 20 μg of Thr or Thr immobilized to SiO₂nanoparticles (2.3 μm) suspended in 100 μl 0.1M PBS (pH=7.4). Theresults are shown in FIG. 2 (>600—Plasma clotting was not observed up to600 seconds, at which time the experiment was terminated).

FIG. 2 demonstrates the storage stability at RT obtained by immobilizingThr to SiO₂ nanoparticles (2.3 μm). For example, the clotting time inplasma of Thr and SiO2˜Thr suspended in PBS and stored at RT for 8 daysincreased by ca. 14 and 1.8, respectively.

Example 6 Clotting Time in Plasma as a Function of Storage at RoomTemperature of Thr and AB˜Thr Suspended in PBS

Clotting time was measured at 37° C. by mixing 50 μl plasma with samplescontaining, each sample, 20 μg of Thr or Thr immobilized to ABnanoparticles suspended in 100 μl 0.1M PBS (pH=7.4). The results areshown in FIG. 3.

FIG. 3 demonstrates the storage stability at RT obtained by immobilizingThr to AB nanoparticles. For example, the clotting time in plasma of Thrand AB˜Thr suspended in PBS and stored at RT for 8 days increased by ca.11 and 2.3, respectively.

Example 7 Clotting Time in Plasma as a Function of Storage Time at RT ofThr and Mag˜Thr Suspended in PBS

Clotting time was measured at 37° C. by mixing 50 μl plasma with samplescontaining, each sample, 20 μg Thr or Thr immobilized to Magnanopartilcles suspended in 100 μl 0.1M PBS (pH=7.4). The results areshown in FIG. 4.

FIG. 4 demonstrates the storage stability at RT obtained by immobilizingThr to Mag nanoparticles. For example, the clotting time in plasma ofThr and Mag˜Thr suspended in PBS and stored at RT for 7 days increasedby ca. 12 and 1.4, respectively.

Example 8 Effect of Sterilization on the Clotting Time in Plasma of Thrand Mag˜Thr Suspended in PBS and Stored at RT

Clotting time was measured at 37° C. by mixing 50 μl plasma with samplescontaining, each sample, 20 μg sterilized or non-sterilized Thr orimmobilized Thr suspended in 100 μl 0.1M PBS (pH=7.4). The results areshown in FIG. 5.

FIG. 5 demonstrates the bacteria stability at RT obtained byimmobilizing Thr to Mag nanoparticles (as shown by sterilizing the Thrand Mag˜Thr suspension in PBS). For example, the clotting time in plasmaof non-sterilized and sterilized Mag˜Thr suspended in PBS and stored atRT for 14 days increased by ca. 1.7 and 1.3, respectively.

Example 9 Effect of Sterilization on the Clotting Time in Blood of Thrand Mag˜Thr Suspended in PBS and Stored at RT

Clotting time was measured at 37° C. by mixing 50 μl blood with samplescontaining, each sample, 20 μg sterilized Thr or immobilized Thrsuspended in 100 μl 0.1M PBS (pH=7.4). The results are shown in FIG. 6.

FIG. 6 demonstrates the bacteria stability at 4° C. and RT obtained byimmobilizing Thr to Mag nanoparticles (as shown by sterilizing the Thrand Mag˜Thr suspension in PBS). For example, the clotting time in bloodof sterilized Thr and Mag˜Thr suspended in PBS and stored at RT for 37days increased by more than 60 and 1.6, respectively.

Example 10 Effect of the ratio [Gelatin]/[Mag] on the Size and on theClotting Time in Plasma and Blood of Lyophilized Mag˜Thr

Clotting time was measured at 37° C. by mixing 50 μl plasma (or blood)with samples containing, each sample, 10 μg of lyophilized immobilizedThr suspended in 100 μl 0.1M PBS (pH=7.4).

For lyophilization, samples containing 10 μg of immobilized Thr anddifferent amounts of gelatin suspended in 100 μl 0.1M PBS were prepared.The samples were then dried by lyophilization. For clottingmeasurements, each dried sample was suspended with 100 μl H₂O. Theresults are shown in the following table. TABLE 4 Effect of the ratio[Gelatin]/[Mag] on the size and on the clotting time in plasma and bloodof lyophilized Mag˜Thr. Clotting Clotting time [Gelatin]/[Mag] Size timein in blood (w/w) (nm) plasma (sec) (sec) 2  69 ± 11 26.6 ± 0.9 21.3 ±1.9 1  68 ± 11 26.4 ± 0.2 18.5 ± 2.4 0.75  78 ± 14 26.6 ± 0.9 18.5 ± 2.20.5  89 ± 19 26.4 ± 0.7 18.5 ± 2.1 0.25 110 ± 21 27.1 ± 0.6 18.8 ± 1.80.05 124 ± 31 28.3 ± 0.6 19.4 ± 1.8 0.01 135 ± 34 34.6 ± 1.1 20.1 ± 1.90 154 ± 37 38.6 ± 1.4 24.1 ± 1.5

Table 4 illustrates a way to dry the Thr immobilized Mag nanoparticleswhile preserving the size and the hemostatic properties of thesenanoparticles after resuspending them. Table 4 illustrates the followinginformation:

1) Drying the Thr immobilized Mag nanoparticles by lyophilization leadsto agglomeration, i.e. the average diameter of the Mag˜Thr nanoparticlessuspended in PBS increased from 70 nm to 154 nm due to lyophilization.Addition of gelatin to the Mag˜Thr nanoparticles suspended in PBS,followed by lyophilization, decreased the size of the agglomeratedMag˜Thr nanoparticles resuspended in PBS. For example, lyophilization ofthe Mag˜Thr nanoparticles suspended in PBS in the presence of increasinggelatin concentration, i.e. at [Gelatin]/[Mag] ratio of 0.25, 0.75 and1, leads, after resuspension in PBS of the dried nanoparticles, to anaverage size of 110, 78 and 68 nm, respectively.

2) The clotting time in plasma and blood of Mag˜Thr nanoparticlessuspended in PBS is preserved by adding to the nanoparticle suspensiongelatin and then drying it by lyophilization.

Similar results to that described in Table 4 for the clotting time inplasma and blood were also obtained for SiO₂˜Thr and AB˜Thr.

Example 11 Effect of Lyophilization in the Absence or Presence ofGelatin on the Clotting Time in Plasma (A) and Blood (B) of Thr andMag˜Thr Suspended in PBS

Clotting time was measured at 37° C. by mixing 50 μl plasma (or blood)with samples containing, each sample, 9.5 μg of lyophilized Thr orimmobilized Thr suspended in 100 μl 0.1M PBS (pH=7.4).

For lyophilization, samples containing, each sample, 9.5 μg of Thr orimmobilized Thr and 0.3 mg gelatin suspended in 100 μl 0.1M PBS wereprepared. The samples were then dried by lyophilization. For clottingmeasurements, each dried sample was suspended with 100 μl H₂O. Theresults are shown in the following table. TABLE 5 Effect oflyophilization in the absence or presence of gelatin on the clottingtime in plasma (A) and blood (B) of Thr and Mag˜Thr suspended in PBS AClotting time in plasma, sec After Thrombin lyophilization conjugatedBefore without nanoparticle lyophilization gelatin Mag˜Thr 31.4 ± 1.945.7 ± 2.1 Thr 11.5 ± 2.1 >600 B Clotting time in blood, sec AfterThrombin conjugated Before lyophilization with nanoparticlelyophilization gelatin Mag˜Thr 22.4 ± 2.1 22.4 ± 2.1 Thr 13.8 ± 2.2 24.8± 2.4

Table 5 demonstrates the effect of gelatin on the lyophilizationstability of Thr and Mag˜Thr nanoparticles suspended in PBS.Lyophilization caused a significant decrease in the clotting time of Thrsuspension in PBS, i.e. in plasma, from 11.5 to >600 sec, and moderatedecrease for Mag˜Thr, i.e. from 31.4 to 45.7 sec. However, the additionof gelatin to the Thr solution in PBS moderated the decrease in theclotting time in plasma of the Thr (from 11.5 to 20.9 sec.), andpreserved the clotting time of the Mag˜Thr nanoparticles suspended inPBS.

A similar effect to that described in Table 5 was also obtained forSiO₂˜Thr and AB˜Thr.

Example 12 Effect of CaCl₂ on the Clotting Time in Plasma and Blood ofLyophilized Thr and Mag˜Thr

Clotting time was measured at 37° C. by mixing 50 μl plasma (or blood)with samples containing 5 μg of lyophilized Thr or immobilized Thrsuspended in 100 μl 0.1M PBS (pH=7.4) in absence or presence of 8.3 mMCaCl₂.

For lyophilization, samples containing 5 μg of Thr or immobilized Thrand 0.27 mg of gelatin suspended in 100 μl 0.1M PBS were prepared. Thesamples were then dried by lyophilization. For clotting measurements,each dried sample was dispersed with 100 μl H₂O in the absence orpresence of 8.3 mM CaCl₂. The results are shown in the following table.TABLE 6 Effect of CaCl₂ on the clotting time in plasma and blood oflyophilized Thr and Mag˜Thr. Clotting time Clotting time Thrombinconjugated CaCl₂ in plasma in blood nanoparticle (mM) (sec) (sec)Mag˜Thr 0 34.6 ± 0.9 23.3 ± 1.3 Mag˜Thr 8.3 15.7 ± 0.4 12.6 ± 1.2 Thr 028.5 ± 0.5 30.6 ± 1.4 Thr 8.3 17.3 ± 0.2 20.9 ± 1.2

Table 6 illustrates the significant effect of CaCl₂, 8.3 mM, on thedecrease in the clotting time in plasma and blood of Thr and Mag˜Thrnanoparticles. For example, the measured clotting times in plasma andblood of resuspended lyophilized Mag˜Thr suspension in PBS in absenceand presence of 8.3 mM CaCl₂ were 34.6 and 23.3 sec., respectively inthe absence of CaCl₂, and 15.7 and 12.6 sec., respectively in thepresence of CaCl₂.

A similar effect to that described in Table 6 was also obtained forSiO₂˜Thr and AB˜Thr.

Example 13 Effect of Thrombin Concentration on the Clotting Time inBlood of Lyophilized Thr and Mag˜Thr in the Presence of 8.3 mM of CaCl₂

Clotting time was measured at 37° C. by mixing 50 μl blood with samplescontaining, each sample, different amounts of lyophilized Thr orimmobilized Thr in 100 μl 0.1M PBS (pH=7.4) in absence or presence of8.3 mM CaCl₂.

For lyophilization, samples containing, each sample, different amountsof Thr or immobilized thrombin and appropriate amounts of gelatinsuspended in 100 μl 0.1M PBS were prepared. The samples were then driedby lyophilization. For clotting measurements, each dried sample wasdispersed with 100 μl H₂O in absence or presence of 8.3 mM CaCl₂. Theresults are shown in FIG. 7.

FIG. 7 also demonstrates the significant effect of 8.3 mM CaCl₂ on thedecrease in the clotting time in blood of various concentrations oflyophilized Thr and Mag˜Thr nanoparticles resuspended in PBS.

A similar effect to that described in FIG. 7 was also obtained forSiO₂˜Thr and AB˜Thr.

Example 14 Effect of Factor XIII on the Clotting Time in Plasma andBlood of Lyophilized Thr and Mag˜Thr

Clotting time was measured at 37° C. by mixing 50 μl plasma (or blood)with samples containing 13 μg of Thr or immobilized lyophilized Thrsuspended in 100 μl 0.1M PBS (pH=7.4).

For lyophilization, samples containing 13 μg of Thr or immobilizedthrombin and 0.35 mg of gelatin suspended in 100 μl 0.1M PBS wereprepared. The samples were then dried by lyophilization. For clottingmeasurements, each dried sample was dispersed with 100 μl H₂O in thepresence of 8.3 mM CaCl₂. The results are shown in the following table.TABLE 7 Effect of Factor XIII on the clotting time in plasma and bloodof lyophilized Thr and Mag˜Thr. [Factor Clotting time Clotting timeXIII] in plasma in blood Bioactive conjugate (μg) (sec) (sec) Mag˜Thr 0 9.2 ± 0.8  8.7 ± 0.2 Mag˜Thr 11.7  7.1 ± 0.7  6.4 ± 0.5 Thr 0 16.3 ±0.4 23.8 ± 0.9 Thr 11.7 16.2 ± 0.3 18.2 ± 0.3

Table 7 demonstrates the effect of factor XIII on the decrease in theclotting time in plasma and blood of Thr and Mag˜Thr nanoparticles. Forexample, the measured clotting times in plasma and blood of resuspendedlyophilized Mag˜Thr suspension in PBS containing 8.3 mM CaCl₂ in theabsence and presence of factor XIII were 9.2 and 7.1 sec., respectivelyin plasma (23% decrease in clotting time), and 8.7 and 6.4 sec.,respectively in blood (24% decrease in clotting time).

A similar effect to that described in Table 7 was also obtained forSiO₂˜Thr and AB˜Thr.

Example 15 In Vivo Trials

Rabbits (female, white, 2.5 Kg, New Zealand) were bled by pricking themain vein of the ear (with 18 G syringes) The bleeding rate wasapproximately 25 μl/sec. Attempts to stop the bleeding were made byplacing (without pressure) a sterile cellulose bandage wetted withlyophilized Mag˜Thr resuspened in PBS in the absence or presence of 8.3mM CaCl₂ upon the wound. Similar trials were performed with cellulosedressings wetted with a Thr PBS solution in the absence or presence of8.3 mM CaCl₂ (similar Thr concentration, 32 μg). Control trials wereaccomplished with cellulose dressings wetted only with PBS in absence orpresence of 8.3 mM CaCl₂. A complete cessation of bleeding was observedafter ca. 45 sec. for both dressings wetted with Thr or lyophilizedMag˜Thr resuspended in PBS, and after ca. 30 sec. with dressings wettedwith Thr or lyophilized Mag˜Thr resuspended in PBS containing 8.3 mMCaCl₂. Conversely, the bleeding of the rabbit treated with the controldressing was not stopped by 2.5 min.; in order to prevent the rabbitfrom dying, the bleeding was ceased by applying strong pressure on thedressing located on the wound.

The above described experiment was performed, for each type of dressing,with 5 rabbits. Similar behavior was observed in each subject.

Similar trials were also performed with gelatin sponges (dressings).Similar behavior was also observed for each subject.

Similar trials to that described above substituting the Mag˜Thrnanoparticles for AB˜Thr were also performed. Similar behavior was alsoobserved for each of the subjects tested.

Example 16 In Vivo Trials

Rabbits (female, white, 2.5 Kg, New Zealand) were bled by pricking themain vein of the ear (with 18 G syringes) The bleeding rate wasapproximately 25 μl/sec. Attempts to stop the bleeding were made byspraying, or dropping, upon the wound a PBS suspension of thelyophilized Mag˜Thr nanoparticles in the absence or presence of 8.3 mMCaCl₂. Then, cellulose (or gelatin) dressings were placed (withoutpressure) upon the wound. Similar trials were performed with a Thr PBSsolution in the absence or presence of 8.3 mM CaCl₂ (similar Thrconcentration, 32 μg) Control trials were accomplished with PBS in theabsence or presence of 8.3 mM CaCl₂. Similar behavior to that indescribed in example 15 was observed.

Similar trials to that described above substituting the Mag˜Thrnanoparticles for AB˜Thr were also performed. Similar behavior was alsoobserved.

Example 17 Controlled Release

Fibrin glue containing antibiotics was prepared by shaking 10 mg oflyophilized Mag˜Thr nanoparticles containing 1 mg antibiotics, i.e.Ampicillin sodium salt, with 0.5 ml of 0.1M PBS (pH=7.4) containing 0.2%fibrinogen for a few minutes at 37° C. The formed fibrin glue in 2 ml of0.1M PBS (pH=7.4) was inserted into a dialysis membrane bag (cut off10,000 m.w.), and then tightly sealed with a closure. The bag wasimmersed in 20 ml 0.1M PBS (pH=7.4) as release medium, and shaken at 37°C. in water bath. A 0.5 ml aliquot was withdrawn at each 0.5 h intervaland an equal volume of fresh PBS was replaced. The concentration ofAmpicillin sodium salt in the dialyzate (determined by UV spectrometryat 226 nm) increased gradually up to 7 h, and about 80% of Ampicillinsodium salt was released within 20 h.

A similar trial was also performed with AB˜Thr nanoparticles containingAmpicillin sodium salt. A similar release profile was also observed.

Example 18 Targeting

Fibrin glue was prepared by shaking 10 mg of lyophilized Mag˜Thrnanoparticles with 0.5 ml of 0.1M PBS (pH=7.4) containing 0.2%fibrinogen for a few minutes at 37 ° C. The formed fibrin glue was thenplaced in 2 ml of 0.1M PBS (pH=7.4). AB˜Thr nanoparticles (1 mg)dispersed in 0.5 ml 0.1M PBS (pH=7.4) were then added to the formedfibrin glue in PBS. The mixture was then shaken at room temperature for1 h. Excess AB˜Thr nanoparticles were then removed from the fibrin glueby 4 centrifugation cycles in PBS. Light microscopy pictures visualizedthe binding of the AB-Thr nanoparticles to the surface of the biologicalglue. Similar behavior was also observed for the SiO₂˜Thr nanoparticles(2.3 μm).

1. Thrombin-conjugated nanoparticles, wherein said nanoparticlescomprise one or more organic and/or inorganic compounds.
 2. Thethrombin-conjugated nanoparticles according to claim 1, wherein thethrombin molecules are covalently-bonded to the surface of thenanoparticles.
 3. The thrombin-conjugated nanoparticles according toclaim 1, wherein the thrombin molecules are covalently-bonded to spacerarms, and wherein said spacer arms are covalently-linked to the surfaceof the nanoparticles.
 4. The thrombin-conjugated nanoparticles accordingto claim 1, wherein the thrombin molecules are physically adsorbed ontospacer arms, and wherein said spacer arms are covalently-linked to thesurface of the nanoparticles.
 5. The thrombin-conjugated nanoparticlesaccording to claim 1, wherein the organic compounds are selected fromthe group consisting of proteins and synthetic polymers, and wherein theinorganic compounds are selected from the group consisting of metaloxides or oxides of metalloids.
 6. The thrombin-conjugated nanoparticlesaccording to claim 5, wherein the nanoparticles are selected from thegroup consisting of magnetic iron oxide-containing nanoparticles,albumin nanoparticles, solid or hollow silica nanoparticles andnanoparticles made of organic polymeric core coated with a silica shell,optionally having magnetic layer interposed between said core and saidsilica shell.
 7. The thrombin-conjugated nanoparticles according toclaim 2, wherein the nanoparticles are magnetic iron oxide-containingnanoparticles having a coating on their surface, and wherein thethrombin molecules are covalently-linked to said coating.
 8. Thethrombin-conjugated nanoparticles according to claim 3, wherein thespacer arm is albumin.
 9. The thrombin-conjugated nanoparticlesaccording to claim 1, further comprising a pharmaceutical agent, whereinsaid pharmaceutical agent is either encapsulated within saidnanoparticles, or bound thereto.
 10. A process, which comprises:providing nanoparticles comprising one or more organic and/or inorganiccompounds, said nanoparticles having reactive chemical groups on theirsurface, and either covalently linking thrombin thereto, or covalentlylinking spacer arms to said reactive chemical groups and subsequentlyallowing thrombin molecules to chemically react with said spacer arms,or to become physically adsorbed thereto, whereby thrombin-conjugatednanoparticles are obtained.
 11. The process according to claim 10,wherein the reactive chemical groups are either activated carbon-carbondouble bonds or aldehyde groups.
 12. The process according to claim 11,which comprises covalently linking thrombin molecules to thenanoparticles by allowing the primary amine groups of the thrombinmolecules to react either with the activated carbon-carbon double bondsvia a Michael addition reaction, or with the aldehyde groups through theformation of Schiff Bases.
 13. The process according to claim 11, whichcomprises covalently linking spacer arms to the nanoparticles byallowing primary amine groups of the spacer arms molecules to reacteither with the activated carbon-carbon double bonds via a Michaeladdition reaction or with the aldehyde groups through the formation ofSchiff Bases, and subsequently allowing primary amine groups of thethrombin molecules to react with carboxyl groups of said spacer arms.14. The process according to claim 10, wherein the spacer arm isalbumin.
 15. A therapeutic composition comprising a therapeuticallyeffective amount of thrombin-conjugated nanoparticles as defined inclaim 1, suitable for use in the preparation of fibrin-based biologicalsealant.
 16. The therapeutic composition according to claim 15, providedin the form of a dry powder comprising thrombin-conjugated nanoparticlesand a dispersant.
 17. The therapeutic composition according to claim 16,wherein the dispersant is gelatin.
 18. A therapeutic compositionaccording to claim 15, provided in the form of a liquid vehiclecomprising thrombin-conjugated nanoparticles and optionally adispersant.
 19. A therapeutic composition according to claim 15, whichfurther comprises one or more additives selected from the groupconsisting of Ca salts, factor XIII and antifibrinolytic agents.
 20. Aprocess for preparing a thrombin formulation provided in the form of adry powder comprising thrombin-conjugated nanoparticles, said processcomprising providing an aqueous suspension of said thrombin-conjugatednanoparticles and drying said aqueous suspension in the presence of asuitable dispersant.
 21. The process according to claim 20, wherein thedrying is accomplished by means of lyophilization.
 22. The processaccording to claim 21, wherein the dispersant is gelatin.
 23. Theprocess according to claim 21, wherein the weight ratio of gelatin tothrombin-conjugated nanoparticles in the aqueous suspension is in therange of 1:2 to 2:1.
 24. The process according to claim 20, wherein theaqueous suspension containing the thrombin-conjugated nanoparticles andthe dispersant is absorbed onto a suitable support, said support beingpreferably in the form of cellulose, collagen or gelatin sheets,following which said support is dried to produce a dry powder ofthrombin-conjugated nanoparticles thereon.
 25. A method for preparingfibrin-containing biological sealant, wherein said method comprisescontacting fibrinogen with a therapeutically effective amount ofthrombin-conjugated nanoparticles as defined in claim 1 in a liquidmedium selected from the group consisting of aqueous solution, plasma orblood; whereby the fibrin sealant is formed.
 26. The method according toclaim 24, which comprises contacting fibrinogen with a dry powdercomprising thrombin-conjugated nanoparticles and a dispersant, or with aliquid suspension of said nanoparticles.
 27. The method according toclaim 25, wherein the dispersant is gelatin.
 28. The method according toclaim 23, wherein the thrombin-conjugated nanoparticles and fibrinogenare contacted in the presence of calcium ions or factor XIII.